Method of producing erythrocytes

ABSTRACT

Provided herein are methods of producing erythrocytes from hematopoietic cells, particularly hematopoietic cells from placental perfusate in combination with hematopoietic cells from umbilical cord blood, wherein the method results in accelerated expansion and differentiation of the hematopoietic cells to more efficiently produce administrable erythrocytes. Further provided herein is a bioreactor in which hematopoietic cell expansion and differentiation takes place.

This application is a divisional of U.S. application Ser. No. 12/187,337, filed Aug. 6, 2008, now U.S. Pat. No. 9,200,253, which claims the benefit of U.S. Provisional Application No. 60/963,894, filed Aug. 6, 2007, each of which is incorporated by reference herein in its entirety.

1. FIELD

Provided herein, generally, are methods of expanding hematopoietic cell populations, e.g., CD34⁺ cell populations, and methods of producing administrable units of erythrocytes from such cell populations. Also provided herein is a bioreactor that accomplishes such expansion and differentiation.

2. BACKGROUND

Each year in the United States approximately 13 million units of blood are used for transfusion or to generate life-saving blood products such as platelets. Voluntary blood donation is utilized by the Red Cross and other agencies to procure from about 500 mL to about 1000 mL whole blood samples. Self-screening of voluntary donation is relatively safe and effective in the US and Western Europe where the incidences of HIV and other adventitious pathogens are relatively low. However, in countries in which HIV and hepatitis are endemic, procurement of safe blood for transfusion can be highly problematic. As an alternative to voluntary blood donation many groups have attempted to develop safe artificial blood substitutes that could undergo long-term storage. While some of these products show significant promise in transiently treating traumatic blood loss, such products are not designed for long-term substitution of red blood cell function. Increasingly there is a need to develop a safe and plentiful supply of erythrocytes that can be administered to patients on the battlefield or civilian hospital settings around the world.

Conventional methods for producing erythrocytes are either inefficient, too small in scale, or too laborious to allow for the continuous, on-site production of erythrocytes. Conventional dish or flask-based culture systems are associated with discontinuous medium exchange, and generally dish-based culture systems cannot be used to handle single batches of >10⁹ cells. A logical further development from dishes are bag technologies, e.g. the Wave Bioreactor, in which the medium volume is significantly enlarged by using bags and cell attachment surface can be enlarged by using buoyant carriers. However, bag-type reactors typically operate from 2×10⁶ to about 6×10⁶ cells/ml medium, requiring significant media dilution during culture and a laborious 10-100 fold debulking. Moreover, bag technologies, and generally all large-vessel stirred tank type bioreactors, do not provide tissue-like physiologic environments that are conducive to “normal” cell expansion and differentiation.

3. SUMMARY

Provided herein are methods of expanding hematopoietic cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells), to differentiating the expanded hematopoietic cells into administrable erythrocytes (red blood cells), and to the production of administrable units of cells comprising the erythrocytes.

In one aspect, provided herein is a method of producing erythrocytes. In one embodiment, the method comprises differentiating hematopoietic cells from human placental perfusate to erythrocytes. In a specific embodiment, the method comprises expanding a population of hematopoietic cells in the absence of a feeder layer, wherein the hematopoietic cells are obtained from human placental perfusate and optionally from human umbilical cord blood; subsequently expanding said hematopoietic cells in the presence of a feeder layer; and differentiating the hematopoietic cells to erythrocytes or progenitors of erythrocytes.

In another aspect, provided herein is a bioreactor for the expansion of hematopoietic cells and differentiation of said hematopoietic cells into erythrocytes. The bioreactor allows for production of a number of erythrocytes equivalent to current methods of producing erythrocytes, in a much smaller volume, by facilitating a continuous erythrocyte production method rather than a batch method. In specific embodiments, the bioreactor comprises a cell culture element, a cell separation element, a gas provision element and/or a medium provision element. In a specific embodiment of the bioreactor, the erythrocytes are collected by magnetic bead separation. In another embodiment of the method, the erythrocytes are collected by partially or fully deoxygenating hemoglobin in said erythrocytes, and attracting the erythrocytes to a surface using a magnetic field.

In another aspect, provided herein is a method of the production of erythrocytes using the bioreactor described herein. In a specific embodiment, provided herein is a method of producing erythrocytes comprising producing erythrocytes using a plurality of the bioreactors disclosed herein. In other specific embodiments of the method, the production of said erythrocytes is automated.

In one aspect, provided herein is a method of producing erythrocytes, comprising (a) expanding a plurality of hematopoietic cells in the absence of feeder cells, optionally in contact with an immunomodulatory compound, wherein the immunomodulatory compound increases the number of hematopoietic cells compared to a plurality of hematopoietic cells not in contact with the immunomodulatory compound, to produce a first expanded hematopoietic cell population; (b) expanding the first expanded hematopoietic cell population in the presence of a plurality of feeder cells to produce a second expanded hematopoietic cell population; (c) contacting said second expanded hematopoietic cell population with one or more factors that cause differentiation of hematopoietic cells in said second expanded hematopoietic cell population into erythrocytes; and (d) isolating said erythrocytes from said second expanded hematopoietic cell population. In a specific embodiment, said isolating of erythrocytes in step (d) is performed continuously. In other specific embodiments, said isolating of erythrocytes in step (d) is performed periodically, e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or more. In another specific embodiment, said isolating of erythrocytes in step (d) is performed periodically when one or more culture condition criteria are met, e.g., achievement in the culture of a particular cell density; achievement in the culture of a particular number of cells per milliliter expressing certain erythrocyte markers, e.g., CD36 or glycophorin A; or the like.

In a specific embodiment, said hematopoietic cells are CD34⁺. In another specific embodiment, said hematopoietic cells are Thy-1⁺, CXCR4⁺, CD133⁺ or KDR⁺. In another specific embodiment, said hematopoietic cells are CD45⁻. In another specific embodiment, said hematopoietic cells are HLA-DR⁻, CD71⁻, CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD19⁻, CD16⁻, CD24⁻, CD56⁻, CD66b⁻ and/or glycophorin A⁻. In another specific embodiment, said hematopoietic stem cells are Lin⁻.

In another specific embodiment, said hematopoietic cells are obtained from cord blood, placental blood, peripheral blood, or bone marrow. In another specific embodiment, said hematopoietic cells are obtained from placental perfusate. In another specific embodiment, said hematopoietic cells are obtained from umbilical cord blood and placental perfusate. In a more specific embodiment, said placental perfusate is obtained by passage of perfusion solution through only the vasculature of a placenta. In another specific embodiment, said hematopoietic cells are human hematopoietic cells.

In another specific embodiment, said feeder cells are from the same individual as the hematopoietic cells. In another specific embodiment, said feeder cells are from a different individual as the hematopoietic cells. In a more specific embodiment, said feeder cells are adherent placental stem cells, bone marrow-derived mesenchymal stem cells, mesenchymal stem cells from peripheral blood, mesenchymal stem cells from cord blood, or stromal stem cells, or a combination of any of the foregoing. In another specific embodiment, said feeder cells are adherent placental stem cells. In a more specific embodiment, said adherent placental stem cells are CD200⁺ or HLA-G⁺; CD73⁺, CD105⁺, and CD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁺; CD73⁺ and CD105⁺ and facilitate the formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said stem cells when said population is cultured under conditions that allow formation of embryoid-like bodies; or OCT-4⁺ and facilitate formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said stem cell when cultured under conditions that allow formation of embryoid-like bodies. In another more specific embodiment, the adherent placental stem cells are CD10⁺, CD34⁻, CD105⁺, and CD200⁺. In another more specific embodiment, the adherent placental stem cells are HLA-A,B,C⁺, CD45⁻, CD133⁻ and CD34⁻. In another more specific embodiment, the adherent placental stem cells are CD10⁺, CD13⁺, CD33⁺, CD45⁻, CD117⁻ and CD133⁻. In another more specific embodiment, the adherent placental stem cells are CD10⁻, CD33⁻, CD44⁺, CD45⁻, and CD117⁻. In another more specific embodiment, the adherent placental stem cells are HLA A,B,C⁺, CD45⁻, CD34⁻, CD133⁻; positive for CD10, CD13, CD38, CD44, CD90, CD105, CD200 and/or HLA-G, and/or negative for CD117. In another more specific embodiment, the adherent placental stem cells are CD200⁺ and CD10⁺, as determined by antibody binding, and CD117⁻, as determined by both antibody binding and RT-PCR. In another more specific embodiment, the adherent placental stem cells are CD10⁺, CD29⁻, CD54⁺, CD200⁺, HLA-G⁺, HLA class 1⁺ and β-2-microglobulin⁺.

In certain embodiments of the method, a plurality of said hematopoietic cells is blood type A, blood type O, blood type AB, blood type O; is Rh positive or Rh negative; blood type M, blood type N, blood type S, or blood type s; blood type P1; blood type Lua, blood type Lub, or blood type Lu(a); blood type K (Kell), k (cellano), Kpa, Kpb, K(a+), Kp(a−b−) or K-k-Kp(a−b−); blood type Le(a−b−), Le(a+b−) or Le(a−b+); blood type Fy a, Fy b or Fy(a−b−); or blood type Jk(a−b−), Jk(a+b−), Jk(a−b+) or Jk(a+b+). In a more specific embodiment, the hematopoietic cells are type O, Rh positive; type O, Rh negative; type A, Rh positive; type A, Rh negative; type B, Rh positive; type B, Rh negative; type AB, Rh positive or type AB, Rh negative. In other specific embodiments of the method, greater than 90%, 95%, 98%, or 99%, or each, of said hematopoietic cells is blood type A, blood type O, blood type AB, blood type O; is Rh positive or Rh negative; blood type M, blood type N, blood type S, or blood type s; blood type P1; blood type Lua, blood type Lub, or blood type Lu(a); blood type K (Kell), k (cellano), Kpa, Kpb, K(a+), Kp(a−b−) or K-k-Kp(a−b−); blood type Le(a−b−), Le(a+b−) or Le(a−b+); blood type Fy a, Fy b or Fy(a−b−); or blood type Jk(a−b−), Jk(a+b−), Jk(a−b+) or Jk(a+b+). In more specific embodiments, the hematopoietic cells are type O, Rh+; type O, Rh negative; type A, Rh positive; type A, Rh negative; type B, Rh positive; type B, Rh negative; type AB, Rh positive or type AB, Rh negative.

As used herein, the term “hematopoietic cells” includes hematopoietic stem cells and hematopoietic progenitor cells, that is, blood cells able to differentiate into erythrocytes.

As used herein, “+”, when used to indicate the presence of a particular cellular marker, means that the cellular marker is detectably present in fluorescence activated cell sorting over an isotype control; or is detectable above background in quantitative or semi-quantitative RT-PCR.

As used herein, “−”, when used to indicate the presence of a particular cellular marker, means that the cellular marker is not detectably present in fluorescence activated cell sorting over an isotype control; or is not detectable above background in quantitative or semi-quantitative RT-PCR.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Flow cytometric analysis of HPP-derived CD34⁺/CD45⁻ and CD34⁺/CD45⁺ cells.

FIGS. 2A-2B: Cell expansion in pomalidomide supplemented IMDM medium. FIG. 2A: Fold expansion of total nucleated cells (TNC). FIG. 2B: Fold expansion of CD34⁺ cells.

FIGS. 3A-3C: CFU-forming activity of expanded cultures. FIG. 3A: Fold expansion of TNC. FIG. 3B: Fold expansion of CD34⁺ cells. FIG. 3C: Expansion of CD34⁺ cells in number of cells. “Compound 1” is pomalidomide.

FIGS. 4A-4B: Ex vivo expansion of CD34⁺ cells from HPP (FIG. 4A) and umbilical cord blood (UCB) (FIG. 4B) in cytokine-supplemented RPMI Medium (see Example 3).

FIGS. 5A-5D: Giemsa staining of CD34⁺ cultures in cytokine supplemented RPMI medium. FIG. 5A: CD34⁺ cells at day 0, showing medium blue color. FIG. 5B: Basophilic normoblasts at day 9, showing medium blue color. FIG. 5C: Orthochromatophilic normoblasts at day 11, showing medium blue color. FIG. 5D: Polychromatophilic erythrocytes/erythrocytes at day 21, showing pink color. All images were obtained at magnification 400×.

5. DETAILED DESCRIPTION

Provided herein is a method of producing erythrocytes from expanded hematopoietic cells, e.g., hematopoietic stem cells and/or hematopoietic progenitor cells. In one embodiment, hematopoietic cells are collected from a source of such cells, e.g., placental perfusate and umbilical cord blood. The hematopoietic cells are expanded first without the use of feeder cells. Such isolation and expansion can be performed in a central facility, which provides expanded hematopoietic cells for shipment to decentralized expansion and differentiation at points of use, e.g., hospital, military base, military front line, or the like. Expansion at point-of-use, in a preferred embodiment, is accomplished using feeder cells. Feeder cell-dependent expansion, according to the methods in Section 5.2.2, below, preferably takes place within a bioreactor, as exemplified herein. Differentiation of erythrocytes, according to the methods of Section 5.3, below, also preferably takes place in the bioreactor. Collection of erythrocytes produced in the method, in a preferred embodiment, is performed continuously or periodically, e.g., during differentiation. The continuous or periodic separation aspect of the method allows for the production of erythrocytes in a substantially smaller space than possible using, e.g., batch methods. The time for collection and expansion of the hematopoietic cells is approximately 5-10 days, typically about 7 days. Erythrocytes are purified on-site and packaged into administrable units.

In one aspect, provided herein is a method of producing erythrocytes, comprising (a) expanding a plurality of hematopoietic cells in the absence of feeder cells, optionally in contact with an immunomodulatory compound, wherein the immunomodulatory compound increases the number of hematopoietic cells compared to a plurality of hematopoietic cells not in contact with the immunomodulatory compound, to produce a first expanded hematopoietic cell population; (b) expanding the first expanded hematopoietic cell population in the presence of a plurality of feeder cells to produce a second expanded hematopoietic cell population; (c) contacting said second expanded hematopoietic cell population with one or more factors that cause differentiation of hematopoietic cells in said second expanded hematopoietic cell population into erythrocytes; and (d) isolating said erythrocytes from said second expanded hematopoietic cell population. In a specific embodiment, said isolating of erythrocytes in step (d) is performed continuously. In other specific embodiments, said isolating of erythrocytes in step (d) is performed periodically, e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or more. In another specific embodiment, said isolating of erythrocytes in step (d) is performed periodically when one or more culture condition criteria are met, e.g., achievement in the culture of a particular cell density; achievement in the culture of a particular number of cells per milliliter expressing certain erythrocyte markers, e.g., CD36 or glycophorin A; or the like.

5.1. Hematopoietic Cells

Hematopoietic cells useful in the methods disclosed herein can be any hematopoietic cells able to differentiate into erythrocytes, e.g., precursor cells, hematopoietic progenitor cells, hematopoietic stem cells, or the like. Hematopoietic cells can be obtained from tissue sources such as, e.g., bone marrow, cord blood, placental blood, peripheral blood, or the like. Hematopoietic cells can be obtained from placenta. In a specific embodiment, the hematopoietic cells are obtained from placental perfusate. Hematopoietic cells from placental perfusate can comprise a mixture of fetal and maternal hematopoietic cells, e.g., a mixture in which maternal cells comprise greater than 5% of the total number of hematopoietic cells. Preferably, hematopoietic cells from placental perfusate comprise at least about 90%, 95%, 98%, 99% or 99.5% fetal cells.

In certain embodiments, the hematopoietic cells are CD34⁺ cells. CD34⁺ hematopoietic cells can, in certain embodiments, express or lack the cellular marker CD38. Thus, in specific embodiments, the hematopoietic cells useful in the methods disclosed herein are CD34⁺CD38⁺ or CD34⁺CD38⁻. In a more specific embodiment, the hematopoietic cell is CD34⁺CD38⁻Lin⁻. In another specific embodiment, the hematopoietic cell is one or more of CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD16⁻, CD19⁻, CD24⁻, CD56⁻, CD66b⁻ and glycophorin A⁻. In another specific embodiment, the hematopoietic cell is CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD16⁻, CD19⁻, CD24⁻, CD56⁻, CD66b⁻ and glycophorin A⁻. In another more specific embodiment, the hematopoietic cell is CD34⁺CD38⁻CD33⁻CD117⁻. In another more specific embodiment, the hematopoietic cell is CD34⁺CD38⁻CD33⁻CD117⁻CD235⁻CD36⁻.

In another embodiment, the hematopoietic cells are CD45⁻. In a specific embodiment, the hematopoietic cells are CD34⁺CD45⁻. In another specific embodiment, the hematopoietic cells are CD34⁺CD45⁺.

In another embodiment, the hematopoietic cell is Thy-1⁺. In a specific embodiment, the hematopoietic cell is CD34⁺Thy-1⁺. In another embodiment, the hematopoietic cells are CD133⁺. In specific embodiments, the hematopoietic cells are CD34⁺CD133⁺ or CD133⁺Thy-1⁺. In another specific embodiment, the CD34⁺ hematopoietic cells are CXCR4⁺. In another specific embodiment, the CD34⁺ hematopoietic cells are CXCR4⁻. In another embodiment, the hematopoietic cells are positive for KDR (vascular growth factor receptor 2). In specific embodiments, the hematopoietic cells are CD34⁺KDR⁺, CD133⁺KDR⁺ or Thy-1⁺KDR⁺. In certain other embodiments, the hematopoietic cells are positive for aldehyde dehydrogenase (ALDH⁺), e.g., the cells are CD34⁺ALDH⁺.

In certain embodiments, the hematopoietic cells are CD34⁻.

The hematopoietic cells can also lack certain markers that indicate lineage commitment, or a lack of developmental naiveté. For example, in another embodiment, the hematopoietic cells are HLA-DR⁻. In specific embodiments, the hematopoietic cells are CD34⁺HLA-DR⁻, CD133⁺HLA-DR⁻, Thy-1⁺HLA-DR⁻ or ALDH⁺HLA-DR⁻ In another embodiment, the hematopoietic cells are negative for one or more, preferably all, of lineage markers CD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b and glycophorin A.

Thus, populations of hematopoietic cells can be selected for use in the methods disclosed herein on the basis of the presence of markers that indicate an undifferentiated state, or on the basis of the absence of lineage markers indicating that at least some lineage differentiation has taken place. Methods of isolating cells on the basis of the presence or absence of specific markers is discussed in detail, e.g., in Section 5.1.2, below.

Hematopoietic cells used in the methods provided herein can be a substantially homogeneous population, e.g., a population comprising at least about 95%, at least about 98% or at least about 99% hematopoietic cells from a single tissue source, or a population comprising hematopoietic cells exhibiting the same hematopoietic cell-associated cellular markers. For example, in various embodiment, the hematopoietic cells can comprise at least about 95%, 98% or 99% hematopoietic cells from bone marrow, cord blood, placental blood, peripheral blood, or placenta, e.g., placenta perfusate.

Hematopoietic cells used in the methods provided herein can be obtained from a single individual, e.g., from a single placenta, or from a plurality of individuals, e.g., can be pooled. Where the hematopoietic cells are obtained from a plurality of individuals and pooled, it is preferred that the hematopoietic cells be obtained from the same tissue source. Thus, in one embodiment, the pooled hematopoietic cells are all from placenta, e.g., placental perfusate, all from placental blood, all from umbilical cord blood, all from peripheral blood, and the like.

Hematopoietic cells used in the methods disclosed herein can comprise hematopoietic cells from two or more tissue sources. Preferably, when hematopoietic cells from two or more sources are combined for use in the methods herein, a plurality of the hematopoietic cells used to produce erythrocytes comprise hematopoietic cells from placenta, e.g., placenta perfusate. In various embodiments, the hematopoietic cells used to produce erythrocytes comprise hematopoietic cells from placenta and from cord blood; from placenta and peripheral blood; form placenta and placental blood, or placenta and bone marrow. In a preferred embodiment, the hematopoietic cells comprise hematopoietic cells from placental perfusate in combination with hematopoietic cells from cord blood, wherein the cord blood and placenta are from the same individual, i.e., wherein the perfusate and cord blood are matched. In embodiments in which the hematopoietic cells comprise hematopoietic cells from two tissue sources, the hematopoietic cells from the sources can be combined in a ratio of, for example, 1:10, 2:9, 3:8, 4:7, 5:6, 6:5, 7:4, 8:3, 9:2, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1.

Preferably, the erythrocytes produced from hematopoietic cells according to the methods provided herein are homogeneous with respect to blood type, e.g., identical with respect to cell surface markers, antigens, or the like. Such homogeneity can be achieved, for example, by obtaining hematopoietic cells from a single individual of the desired blood type. In embodiments in which hematopoietic cells are pooled from a plurality of individuals, it is preferred that each of the individuals shares at least one, at least two, or at least three or more antigenic blood determinants in common. In various embodiments, for example, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, blood type O, blood type A, blood type B, or blood type AB. In other embodiments, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, Rh positive, or Rh negative. In a specific embodiment, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, O positive and Rh negative. In more specific embodiments, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, O positive, O negative, A positive, A negative, B positive, B negative, AB positive, or AB negative. In other specific embodiments, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, blood type M, blood type N, blood type S, or blood type s. In other specific embodiments, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, blood type P1. In other specific embodiments, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, blood type Lua, blood type Lub, or blood type Lu(a). In other specific embodiments, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, blood type K (Kell), k (cellano), Kpa, Kpb, K(a+), Kp(a−b−) or K-k-Kp(a−b−). In other specific embodiments, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, blood type Le(a−b−), Le(a+b−) or Le(a−b+). In other specific embodiments, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, blood type Fy a, Fy b or Fy(a−b−). In other specific embodiments, the individual from which the hematopoietic cells are obtained is, or each of the individuals from which hematopoietic cells are obtained are, blood type Jk(a−b−), Jk(a+b−), Jk(a−b+) or Jk(a+b+). In other specific embodiments, the individual from whom the hematopoietic cells are obtained is classifiable within blood group Diego, Cartwright, Xgm Scianna, Bombrock, Colton, Lansteiner-Weiner, Chido/Rogers, Hh, Kx, Gergich, Cromer, Knops, Indian, Ok, Raph, or JMH. In other specific embodiments, each of the individuals from which hematopoietic cells are obtained are of the same blood type within a blood typing system or group of antigenic determinants, wherein said blood typing system or group of antigenic determinants are Diego, Cartwright, Xgm Scianna, Bombrock, Colton, Lansteiner-Weiner, Chido/Rogers, Hh, Kx, Gergich, Cromer, Knops, Indian, Ok, Raph, or JMH.

5.1.1. Placental Hematopoietic Stem Cells

In certain embodiment, the hematopoietic cells used in the methods provided herein are placental hematopoietic cells. As used herein, “placental hematopoietic cells” means hematopoietic cells obtained from the placenta itself, and not from placental blood or from umbilical cord blood. In one embodiment, placental hematopoietic cells are CD34⁺. In a specific embodiment, the placental hematopoietic cells are predominantly (e.g., at least about 90%, 95% or 98%) CD34⁺CD38⁻ cells. In another specific embodiment, the placental hematopoietic cells are predominantly (e.g., at least about 90%, 95% or 98%) CD34⁺CD38⁺ cells. Placental hematopoietic cells can be obtained from a post-partum mammalian (e.g., human) placenta by any means known to those of skill in the art, e.g., by perfusion.

In another embodiment, the placental hematopoietic cell is CD45⁻. In a specific embodiment, the hematopoietic cell is CD34⁺CD45⁻. In another specific embodiment, the placental hematopoietic cells are CD34⁺CD45⁺.

5.1.1.1. Obtaining Placental Hematopoietic Cells by Perfusion

Placental hematopoietic cells can be obtained using perfusion. Methods of perfusing mammalian placenta to obtain cells, including placental hematopoietic cells, are disclosed, e.g., in U.S. Pat. No. 7,045,148, entitled “Method of Collecting placental Stem Cells,” U.S. Pat. No. 7,255,879, entitled “Post-Partum Mammalian Placenta, Its Use and Placental Stem Cells Therefrom,” and in U.S. Application No. 2007/0190042, entitled “Improved Medium for Collecting Placental Stem Cells and Preserving Organs,” the disclosures of which are hereby incorporated by reference in their entireties.

Placental hematopoietic cells can be collected by perfusion, e.g., through the placental vasculature, using, e.g., a saline solution (for example, phosphate-buffered saline, a 0.9% NaCl solution, or the like), culture medium or organ preservation solution as a perfusion solution. In one embodiment, a mammalian placenta is perfused by passage of perfusion solution through either or both of the umbilical artery and umbilical vein. The flow of perfusion solution through the placenta may be accomplished using, e.g., gravity flow into the placenta. Preferably, the perfusion solution is forced through the placenta using a pump, e.g., a peristaltic pump. The umbilical vein can be, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula, that is connected to a sterile connection apparatus, such as sterile tubing, which, in turn is connected to a perfusion manifold.

In preparation for perfusion, the placenta is preferably oriented (e.g., suspended) in such a manner that the umbilical artery and umbilical vein are located at the highest point of the placenta. The placenta can be perfused by passage of a perfusion fluid through the placental vasculature and surrounding tissue. The placenta can also be perfused by passage of a perfusion fluid into the umbilical vein and collection from the umbilical arteries, or passage of a perfusion fluid into the umbilical arteries and collection from the umbilical vein.

In one embodiment, for example, the umbilical artery and the umbilical vein are connected simultaneously, e.g., to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution. The perfusion solution is passed into the umbilical vein and artery. The perfusion solution exudes from and/or passes through the walls of the blood vessels into the surrounding tissues of the placenta, and is collected in a suitable open vessel, e.g., a sterile pan, from the surface of the placenta that was attached to the uterus of the mother during gestation. The perfusion solution may also be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in the wall of the placenta which interfaced with the maternal uterine wall. Placental cells that are collected by this method, which can be referred to as a “pan” method, are typically a mixture of fetal and maternal cells.

In another embodiment, the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or is passed through the umbilical artery and collected from the umbilical veins. Placental cells collected by this method, which can be referred to as a “closed circuit” method, are typically almost exclusively fetal.

The closed circuit perfusion method can, in one embodiment, be performed as follows. A post-partum placenta is obtained within about 48 hours after birth. The umbilical cord is clamped and cut above the clamp. The umbilical cord can be discarded, or can processed to recover, e.g., umbilical cord stem cells, and/or to process the umbilical cord membrane for the production of a biomaterial. The amniotic membrane can be retained during perfusion, or can be separated from the chorion, e.g., using blunt dissection with the fingers. If the amniotic membrane is separated from the chorion prior to perfusion, it can be, e.g., discarded, or processed, e.g., to obtain stem cells by enzymatic digestion, or to produce, e.g., an amniotic membrane biomaterial, e.g., the biomaterial described in U.S. Application Publication No. 2004/0048796.

After cleaning the placenta of all visible blood clots and residual blood, e.g., using sterile gauze, the umbilical cord vessels are exposed, e.g., by partially cutting the umbilical cord membrane to expose a cross-section of the cord. The vessels are identified, and opened, e.g., by advancing a closed alligator clamp through the cut end of each vessel. The apparatus, e.g., plastic tubing connected to a perfusion device or peristaltic pump, is then inserted into each of the placental arteries. The pump can be any pump suitable for the purpose, e.g., a peristaltic pump. Plastic tubing, connected to a sterile collection reservoir, e.g., a blood bag such as a 250 mL collection bag, is then inserted into the placental vein. Alternatively, the tubing connected to the pump is inserted into the placental vein, and tubes to a collection reservoir(s) are inserted into one or both of the placental arteries. The placenta is then perfused with a volume of perfusion solution, e.g., about 750 ml of perfusion solution. Cells in the perfusate are then collected, e.g., by centrifugation.

In one embodiment, the proximal umbilical cord is clamped during perfusion, and more preferably, is clamped within 4-5 cm (centimeter) of the cord's insertion into the placental disc.

The first collection of perfusion fluid from a mammalian placenta during the exsanguination process is generally colored with residual red blood cells of the cord blood and/or placental blood. The perfusion fluid becomes more colorless as perfusion proceeds and the residual cord blood cells are washed out of the placenta. Generally from 30 to 100 ml (milliliter) of perfusion fluid is adequate to initially exsanguinate the placenta, but more or less perfusion fluid may be used depending on the observed results.

The volume of perfusion liquid used to collect placental hematopoietic cells may vary depending upon the number of hematopoietic cells to be collected, the size of the placenta, the number of collections to be made from a single placenta, etc. In various embodiments, the volume of perfusion liquid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL. Typically, the placenta is perfused with 700-800 mL of perfusion liquid following exsanguination.

The placenta can be perfused a plurality of times over the course of several hours or several days to obtain placental hematopoietic cells. Where the placenta is to be perfused a plurality of times, it may be maintained or cultured under aseptic conditions in a container or other suitable vessel, and perfused with a stem cell collection composition (see U.S. Application Publication No. 2007/0190042, the disclosure of which is incorporated herein by reference in its entirety), or a standard perfusion solution (e.g., a normal saline solution such as phosphate buffered saline (“PBS”)) with or without an anticoagulant (e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin), and/or with or without an antimicrobial agent (e.g., β-mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g., at 40-100 μg/ml), penicillin (e.g., at 40 U/ml), amphotericin B (e.g., at 0.5 μg/ml). In one embodiment, an isolated placenta is maintained or cultured for a period of time without collecting the perfusate, such that the placenta is maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or more days before perfusion and collection of perfusate. The perfused placenta can be maintained for one or more additional time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused a second time with, e.g., 700-800 mL perfusion fluid. The placenta can be perfused 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment, perfusion of the placenta and collection of perfusion solution, e.g., stem cell collection composition, is repeated until the number of recovered nucleated cells falls below 100 cells/ml. The perfusates at different time points can be further processed individually to recover time-dependent populations of cells, e.g., placental hematopoietic cells. Perfusates from different time points can also be pooled.

5.1.1.2. Obtaining Placental Hematopoietic Cells by Tissue Disruption

Hematopoietic cells can be isolated from placenta by perfusion with a solution comprising one or more proteases or other tissue-disruptive enzymes (e.g., trypsin, collagenase, papain, chymotrypsin, subtilisin, hyaluronidase; a cathepsin, a caspase, a calpain, chymosin, plasmepsin, pepsin, or the like). In a specific embodiment, a placenta or portion thereof (e.g., amniotic membrane, amnion and chorion, placental lobule or cotyledon, umbilical cord, or combination of any of the foregoing) is brought to 25-37° C., and is incubated with one or more tissue-disruptive enzymes in 200 mL of a culture medium for 30 minutes. Cells from the perfusate are collected, brought to 4° C., and washed with a cold inhibitor mix comprising 5 mM EDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol. The stem cells are washed after several minutes with cold (e.g., 4° C.) stem cell collection composition.

In one embodiment, the placenta can be disrupted mechanically (e.g., by crushing, blending, dicing, mincing or the like) to obtain the hematopoietic cells. The placenta can be used whole, or can be dissected into components prior to physical disruption and/or enzymatic digestion and hematopoietic cell recovery. For example, hematopoietic cells can be obtained from the amniotic membrane, chorion, umbilical cord, placental cotyledons, or any combination thereof.

Placental hematopoietic cells can also be obtained by enzymatic disruption of the placenta using a tissue-disrupting enzyme, e.g., trypsin, collagenase, papain, chymotrypsin, subtilisin, hyaluronidase; a cathepsin, a caspase, a calpain, chymosin, plasmepsin, pepsin, or the like. Enzymatic digestion preferably uses a combination of enzymes, e.g., a combination of a matrix metalloprotease and a neutral protease, for example, a combination of collagenase and dispase. In one embodiment, enzymatic digestion of placental tissue uses a combination of a matrix metalloprotease, a neutral protease, and a mucolytic enzyme for digestion of hyaluronic acid, such as a combination of collagenase, dispase, and hyaluronidase or a combination of LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.) and hyaluronidase. Other enzymes that can be used to disrupt placenta tissue include papain, deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, or elastase. Serine proteases may be inhibited by alpha 2 microglobulin in serum and therefore the medium used for digestion is usually serum-free. EDTA and DNase are commonly used in enzyme digestion procedures to increase the efficiency of cell recovery. The digestate is preferably diluted so as to avoid trapping stem cells within the viscous digest.

Any combination of tissue digestion enzymes can be used. Typical concentrations for tissue digestion enzymes include, e.g., 50-200 U/mL for collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100 U/mL for elastase. Proteases can be used in combination, that is, two or more proteases in the same digestion reaction, or can be used sequentially in order to liberate placental stem cells. For example, in one embodiment, a placenta, or part thereof, is digested first with an appropriate amount of collagenase I at 2 mg/ml for 30 minutes, followed by digestion with trypsin, 0.25%, for 10 minutes, at 37° C. Serine proteases are preferably used consecutively following use of other enzymes.

In another embodiment, the tissue can further be disrupted by the addition of a chelator, e.g., ethylene glycol bis(2-aminoethyl ether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA) to the stem cell collection composition comprising the stem cells, or to a solution in which the tissue is disrupted and/or digested prior to isolation of the placental hematopoietic cells.

It will be appreciated that where an entire placenta, or portion of a placenta comprising both fetal and maternal cells (for example, where the portion of the placenta comprises the chorion or cotyledons), the placental hematopoietic cells collected will comprise a mix of placental stem cells derived from both fetal and maternal sources. Where a portion of the placenta that comprises no, or a negligible number of, maternal cells (for example, amnion), the placental stem cells collected will comprise almost exclusively fetal placental stem cells.

5.1.2. Isolation, Sorting, and Characterization of Cells

Cells, including hematopoietic cells from any source, e.g., mammalian placenta, can initially be purified from (i.e., be isolated from) other cells by Ficoll gradient centrifugation. Such centrifugation can follow any standard protocol for centrifugation speed, etc. In one embodiment, for example, cells collected from the placenta are recovered from perfusate by centrifugation at 5000× g for 15 minutes at room temperature, which separates cells from, e.g., contaminating debris and platelets. In another embodiment, placental perfusate is concentrated to about 200 ml, gently layered over Ficoll, and centrifuged at about 1100× g for 20 minutes at 22° C., and the low-density interface layer of cells is collected for further processing.

Cell pellets can be resuspended in, e.g., fresh saline solution, or a medium suitable for stem cell maintenance, e.g., IMDM serum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL, NY). The total mononuclear cell fraction can be isolated, e.g., using LYMPHOPREP® (Nycomed Pharma, Oslo, Norway) according to the manufacturer's recommended procedure.

As used herein, “isolating” cells, including placental cells, e.g., placental hematopoietic cells or placental stem cells, means to remove at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cells with which the isolated cells are normally associated in the intact tissue, e.g., mammalian placenta. A cell from an organ is “isolated” when the cell is present in a population of cells that comprises fewer than 50% of the cells with which the stem cell is normally associated in the intact organ.

Adherent placental cells obtained by perfusion or digestion, e.g., for use as feeder cells, can, for example, be further, or initially, isolated by differential trypsinization using, e.g., a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis Mo.). Differential trypsinization is possible because placental stem cells typically detach from plastic surfaces within about five minutes whereas other adherent populations typically require more than 20-30 minutes incubation. The detached placental stem cells can be harvested following trypsinization and trypsin neutralization, using, e.g., Trypsin Neutralizing Solution (TNS, Cambrex). In one embodiment of isolation of adherent cells, aliquots of, for example, about 5-10×10⁶ cells are placed in each of several T-75 flasks, preferably fibronectin-coated T75 flasks. In such an embodiment, the cells can be cultured with commercially available Mesenchymal Stem Cell Growth Medium (MSCGM) (Cambrex), and placed in a tissue culture incubator (37° C., 5% CO₂). After 10 to 15 days, non-adherent cells are removed from the flasks by washing with PBS. The PBS is then replaced by MSCGM. Flasks are preferably examined daily for the presence of various adherent cell types and in particular, for identification and expansion of clusters of fibroblastoid cells.

The number and type of cells collected from a mammalian placenta can be monitored, for example, by measuring changes in morphology and cell surface markers using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., staining with tissue specific or cell-marker specific antibodies) fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), by examination of the morphology of cells using light or confocal microscopy, and/or by measuring changes in gene expression using techniques well known in the art, such as PCR and gene expression profiling. These techniques can be used, too, to identify cells that are positive for one or more particular markers. For example, using antibodies to CD34, one can determine, using the techniques above, whether a cell comprises a detectable amount of CD34, in an assay such as an ELISA or RIA, or by FACS; if so, the cell is CD34⁺. Likewise, if a cell, e.g., a feeder cell, e.g., an adherent placental stem cell, produces enough OCT-4 RNA to be detectable by RT-PCR, or significantly more OCT-4 RNA than an adult cell, the cell is OCT-4⁺. Antibodies to cell surface markers (e.g., CD markers such as CD34) and the sequence of stem cell-specific genes, such as OCT-4, are well-known in the art.

Placental cells, particularly cells that have been isolated by Ficoll separation, differential adherence, or a combination of both, may be sorted using fluorescence activated cell sorting (FACS). FACS is a well-known method for separating particles, including cells, based on the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laser excitation of fluorescent moieties in the individual particles results in a small electrical charge allowing electromagnetic separation of positive and negative particles from a mixture. In one embodiment, cell surface marker-specific antibodies or ligands are labeled with distinct fluorescent labels. Cells are processed through the cell sorter, allowing separation of cells based on their ability to bind to the antibodies used. FACS sorted particles may be directly deposited into individual wells of 96-well or 384-well plates to facilitate separation and cloning.

In one embodiment, stem cells from placenta are sorted, e.g., isolated, on the basis of expression one or more of the markers CD34, CD38, CD44, CD45, CD73, CD105, CD117, CD200, OCT-4 and/or HLA-G.

In another embodiment, hematopoietic cells, e.g., CD34⁺, CD133⁺, KDR⁺ or Thy-1⁺ cells, are sorted, e.g., isolated, on the basis of markers characteristic of undifferentiated hematopoietic cells. Such sorting can be done, e.g., in a population of cells that has not been sorted, e.g., a population of cells from a perfusion or a tissue digestion, wherein CD34⁺ cells represent a minority of the cells present in the population. Such sorting can also be done in a population of cells that is mostly (e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%) hematopoietic cells as, for example, a purification step. For example, in a specific embodiment, CD34⁺ cells, KDR⁺ cells, Thy-1⁺ cells, and/or CD133⁺ cells are retained during sorting to produce a population of undifferentiated hematopoietic cells.

In another embodiment, cells, e.g., hematopoietic cells are sorted, e.g., excluded, on the basis of markers of lineage-differentiated cells. For example, cells, in a population of hematopoietic cells, that are CD2⁺, CD3⁺, CD11b⁺, CD11c⁺, CD14⁺, CD16⁺, CD19⁺, CD24⁺, CD56⁺, CD66b⁺ and/or glycophorin A⁺ are excluded during sorting from the population of hematopoietic cells to produce a population of undifferentiated hematopoietic cells.

In another embodiment, hematopoietic cells can be sorted, e.g., isolated, on the basis of lack of expression of, e.g., lineage markers. In a specific embodiment, for example, hematopoietic cells, e.g., CD34⁺ cells, can be isolated based on a determination that the cells are one or more of CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD16⁻, CD19⁻, CD24⁻, CD56⁻, CD66b⁻ and/or glycophorin A⁻.

In another embodiment, cells, e.g., adherent placental stem cells, are sorted first on the basis of their expression of CD34, wherein CD34⁻ cells are retained. In a specific embodiment, CD34⁻ cells that are CD200⁺HLA-G⁺ are separated from all other CD34⁻ cells. In another embodiment, cells from placenta are based on their expression of markers CD200 and/or HLA-G; for example, cells displaying either of these markers are isolated for further use. Cells that express, e.g., CD200 and/or HLA-G can, in a specific embodiment, be further sorted based on their expression of CD73 and/or CD105, or epitopes recognized by antibodies SH2, SH3 or SH4, or lack of expression of CD34, CD38 or CD45. For example, in one embodiment, placental cells are sorted by expression, or lack thereof, of CD200, HLA-G, CD73, CD105, CD34, CD38 and CD45, and placental cells that are CD200⁺, HLA-G⁺, CD73⁺, CD105⁺, CD34⁻, CD38⁻ and CD45⁻ are isolated from other placental cells for further use.

In another embodiment, magnetic beads can be used to separate cells, e.g., DYNABEADS® (Invitrogen). The cells may be sorted using a magnetic activated cell sorting (MACS) technique, a method for separating particles based on their ability to bind magnetic beads (0.5-100 μm diameter). A variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody that specifically recognizes a particular cell surface molecule or hapten. The beads are then mixed with the cells to allow binding. Cells are then passed through a magnetic field to separate out cells having the specific cell surface marker. In one embodiment, these cells can then isolated and re-mixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells are again passed through a magnetic field, isolating cells that bound both the antibodies. Such cells can then be diluted into separate dishes, such as microtiter dishes for clonal isolation.

Adherent placental stem cells, e.g., to be used as feeder cells, can also be characterized and/or sorted based on cell morphology and growth characteristics. For example, placental stem cells can be characterized as having, and/or selected on the basis of, e.g., a fibroblastoid appearance in culture. Placental stem cells can also be characterized as having, and/or be selected, on the basis of their ability to form embryoid-like bodies. In one embodiment, for example, placental cells that are fibroblastoid in shape, express CD73 and CD105, and produce one or more embryoid-like bodies in culture are isolated from other placental cells. In another embodiment, OCT-4⁺ placental cells that produce one or more embryoid-like bodies in culture are isolated from other placental cells.

In another embodiment, placental stem cells, e.g., placental hematopoietic cells or adherent placental stem cells, can be identified and characterized by a colony forming unit assay. Colony forming unit assays are commonly known in the art, such as MESEN CULT™ medium (Stem Cell Technologies, Inc., Vancouver British Columbia)

Placental stem cells can be assessed for viability, proliferation potential, and longevity using standard techniques known in the art, such as trypan blue exclusion assay, fluorescein diacetate uptake assay, propidium iodide uptake assay (to assess viability); and thymidine uptake assay, MTT cell proliferation assay (to assess proliferation). Longevity may be determined by methods well known in the art, such as by determining the maximum number of population doubling in an extended culture.

Placental stem cells can also be separated from other placental cells using other techniques known in the art, e.g., selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection); separation based upon differential cell agglutinability in the mixed population as, for example, with soybean agglutinin; freeze-thaw procedures; filtration; conventional and zonal centrifugation; centrifugal elutriation (counter-streaming centrifugation); unit gravity separation; countercurrent distribution; electrophoresis; and the like.

5.2. Expansion of Hematopoietic Cells

Once a population of hematopoietic cells is obtained, the population is expanded. One unit of erythrocytes is expected to comprise about 1-2×10¹² red blood cells. Hematopoietic stem cell population doubling requires approximately 36 hours. Thus, starting from about 5×10⁷ hematopoietic cells according to standard methods, and assuming 100% efficiency in expansion and differentiation, production of a unit of erythrocytes would require approximately 14 hematopoietic cell population doublings, or approximately 3 weeks. The method described in detail below improves on standard methods by improving the culture conditions of hematopoietic cells and increasing the number of hematopoietic cells during expansion per unit time.

5.2.1. Shortened Hematopoietic Cell Expansion Time

Cells, including hematopoietic cells, comprise cell cycle control mechanisms, which include cyclins and cyclin-dependent kinases (CDKs), that control the rate of cell division. Cell cycle checkpoints are used by cells to monitor and regulate the progress of the cell cycle. If a cell fails to meet the requirements of a phase it will not be allowed to proceed to the next phase until the requirements have been met. The processes associated with qualifying the cell for progression through the different phases of the cell cycle (checkpoint regulation) are relatively slow and contribute to the relatively modest rate of cell division observed in mammalian cells, even under optimal in vitro culture conditions.

In one embodiment of the method of producing erythrocytes, the method uses hematopoietic cells that have a reduced population doubling time. In a specific embodiment, the hematopoietic cells are modified to express higher-than-normal levels of a cell cycle activator, or a lower-than-normal level of a cell cycle inhibitor, wherein the engineered cells have a detectably shorter doubling time than unmodified hematopoietic cells. In a more specific embodiment, the hematopoietic cells are modified to express a higher-than-normal level of one or more of the cell cycle activator cyclin T2 (CCNT2), cyclin T2B (CCNT2B), CDC7L1, CCN1, cyclin G (CCNG2), cyclin H (CCNH), CDKN2C, CDKN2D, CDK4, cyclin D1, cyclin A, cyclin B, Hes1, Hox genes and/or FoxO.

In another more specific embodiment, the hematopoietic cells express a lower-than-normal level of CDK inhibitors p21, p27 and/or TReP-132. Reduction of expression of CDK inhibitors can be accomplished by any means known in the art, e.g., the use of small molecule inhibitors, antisense oligonucleotides targeted to a p21, p27 and/or TReP-132 DNA, pre-mRNA or mRNA sequence, RNAi, or the like.

Modifications of hematopoietic progenitor cells in the context of the present method of producing erythrocytes are expected to be safe in a therapeutic context, as erythrocytes are enucleated and incapable of replication.

In another specific embodiment, the hematopoietic cells are modified to express higher-than-normal levels of a cell cycle activator, wherein the engineered cells have a detectably shorter doubling time than, or detectably increased rate of proliferation compared to, unmodified hematopoietic cells, and where the increased expression of a cell cycle activator is inducible. Any inducible promoter known in the art can be used to construct such a modified hematopoietic cell, e.g., a tetracycline-inducible gene expression system using a stably expressed reverse tetracycline-controlled transactivator (rtTA) under the control of a CMV promoter (e.g., REVTET-ON® System, Clontech Laboratories, Palo Alto, Calif.); U.S. Patent Application Publication No. 2007/0166366 “Autologous Upregulation Mechanism Allowing Optimized Cell Type-Specific and Regulated Gene Expression Cells”; and U.S. Patent Application Publication No. 2007/0122880 “Vector for the Inducible Expression of Gene Sequences,” the disclosure of each of which is incorporated herein by reference in its entirety.

Expression of a gene encoding a cell cycle inhibitor or negative cell cycle regulator can be disrupted in a hematopoietic cell, e.g., by homologous or non-homologous recombination using standard methods. Disruption of expression of a cell cycle inhibitor or negative cell cycle regulator can also be effected, e.g., using an antisense molecule to, e.g., p21, p27 and/or TReP-132.

In another embodiment, hematopoietic cells used to produce erythrocytes are modified to express notch 1 ligand such that expression of the notch 1 ligand results in detectably decreased senescence of the hematopoietic cells compared to unmodified hematopoietic cells; see Berstein et al., U.S. Patent Application Publication 2004/0067583 “Methods for Immortalizing Cells,” the disclosure of which is incorporated herein by reference in its entirety.

In another specific embodiment, the medium in which the hematopoietic cells are expanded enhance faithful DNA replication, e.g., the medium includes one or more antioxidants.

In a preferred embodiment, the method of producing erythrocytes includes a step that excludes any modified hematopoietic cells, or pre-erythrocyte precursors, from the final population of isolated erythrocytes produced in the method disclosed herein. Such separation can be accomplished as described elsewhere herein on the basis of one or more markers characteristic of hematopoietic cells not fully differentiated into erythrocytes. The exclusion step can be performed subsequent to an isolation step in which erythrocytes are selected on the basis of erythrocyte-specific markers, e.g., CD36 and/or glycophorin A.

5.2.2. Feeder Cell-Independent Expansion of Hematopoietic Cells

In certain embodiments, hematopoietic cells used in the methods provided herein are expanded in culture without the use of a feeder layer to produce a population of expanded hematopoietic cells to produce, e.g., a first expanded hematopoietic cell population. The hematopoietic cells can be expanded by any feeder cell-independent method known to those of skill in the art. In one embodiment, feeder-free expansion of hematopoietic cells is the first of at least two expansion steps prior to differentiation of the hematopoietic cells into erythrocytes.

Feeder cell-independent expansion of hematopoietic cells can take place in any container compatible with cell culture and expansion, e.g., flask, tube, beaker, dish, multiwell plate, bag or the like. In a specific embodiment, feeder cell-independent expansion of hematopoietic cells takes place in a bag, e.g., a flexible, gas-permeable fluorocarbon culture bag (for example, from American Fluoroseal). In a specific embodiment, the container in which the hematopoietic cells are expanded is suitable for shipping, e.g., to a site such as a hospital or military zone wherein the expanded hematopoietic cells are further expanded and differentiated, e.g., using the bioreactor described below.

Hematopoietic cells, in certain embodiments, are expanded in a culture medium comprising one or more cytokines or growth factors. In one embodiment, the medium in which the hematopoietic cells are expanded comprise one or more of Flt-3 ligand, thrombopoietin, and stem cell factor (SCF). In a specific embodiment, hematopoietic cells at about 2×10⁴ cells per milliliter are expanded in contact with about 50 ng/mL Flt-3 ligand, about 100 ng/mL thrombopoietin, and about 100 ng/mL SCF. Expansion times can range, e.g., from about 3 days to about 21 days, e.g., about 7 days.

In one embodiment, hematopoietic cells are expanded by culturing said cells in contact with an immunomodulatory compound, e.g., a TNF-α inhibitory compound, for a time and in an amount sufficient to cause a detectable increase in the proliferation of the hematopoietic cells over a given time, compared to an equivalent number of hematopoietic cells not contacted with the immunomodulatory compound. See, e.g., U.S. Patent Application Publication No. 2003/0235909, the disclosure of which is hereby incorporated by reference in its entirety. In a preferred embodiment, the immunomodulatory compound is 3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione; 3-(4′aminoisolindoline-1′-one)-1-piperidine-2,6-dione; 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; 4-amino-2-[(3RS)-2,6-dioxopiperidin-3-yl]-2H-isoindole-1,3-dione; α-(3-aminophthalimido) glutarimide; pomalidomide, lenalidomide, or thalidomide. In another embodiment, said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O, the other of X and Y is C═O or CH₂, and R² is hydrogen or lower alkyl, or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. In another embodiment, said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴, (C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³, C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, (C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or (C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or (C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or (C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join to form a heterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. In another embodiment, said immunomodulatory compound is a compound having the structure

wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R², R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has a value of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene, pentamethylene, hexamethylene, or —CH₂CH₂X₁CH₂CH₂— in which X₁ is —O—, —S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In a specific, preferred embodiment, expansion of hematopoietic cells is performed in IMDM supplemented with 20% BITS (BSA, recombinant human insulin and transferrin), SCF, Flt-3 ligand, IL-3, and 4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (10 μM in 0.05% DMSO). In a more specific embodiment, about 5×10⁷ hematopoietic cells, e.g., CD34⁺ cells, are expanded in the medium to from about 5×10¹⁰ cells to about 5×10¹² cells, which are resuspended in 100 mL of IMDM to produce a population of expanded hematopoietic cells. The population of expanded hematopoietic cells is preferably cryopreserved to facilitate shipping.

The expanded hematopoietic cells produced without feeder cells as described above, e.g., the first expanded hematopoietic cell population, can be further expanded using feeder cells to produce a second expanded hematopoietic cell population, as described in the following Section.

5.2.3. Expansion Using Feeder Layers

In another embodiment, the hematopoietic cells used in the methods provided herein, e.g., a first expanded hematopoietic cell population produced by non-feeder cell-dependent expansion of hematopoietic cells as described above, can be cultured and expanded using a layer of feeder cells, e.g., to produce a second expanded hematopoietic cell population. In one embodiment, expansion of the hematopoietic cells in the presence of a feeder cell layer is the second of at least two hematopoietic cell expansion steps prior to differentiation of the hematopoietic cells to erythrocytes. Though not wishing to be bound by theory, expansion with feeder cells is expected to be “asymmetric,” that is, expansion produces a combination of expansion of relatively undifferentiated hematopoietic cells and relatively more differentiated, lineage-committed cells. Lineage-committed cells can, in certain embodiments, be removed during the expansion phase.

The feeder cells can be any feeder cells used or known to be useful in the art. Feeder cells can be of the same species as the hematopoietic cells, or of a different species. In certain embodiments, feeder cells are from the same individual as the hematopoietic cells, e.g., the feeder cells and hematopoietic cells are matched. Feeder cells, like the hematopoietic cells, can be autologous to a particular recipient. Feeder cell types useful for culturing hematopoietic cells can be, e.g., bone marrow-derived mesenchymal stem cells (e.g., see U.S. Pat. No. 5,486,359), tissue culture plastic-adherent placental stem cells, mesenchymal-like stem cells from cord blood, placental blood or peripheral blood, adult stem cells, or the like). In another embodiment, the feeder cells are fully-differentiated cells, e.g., fibroblasts, such as human skin fibroblasts or mouse embryonic fibroblasts, or human umbilical vein endothelial cells.

In certain embodiments, the feeder cells are not treated to reduce proliferation or differentiation. In certain other embodiments, the feeder layer cells are treated with, e.g., mitomycin C or ionizing radiation, e.g., gamma radiation, to suppress or prevent proliferation of the feeder cells, e.g., about 100 cGy to about 100 Gray, e.g., 1 Gray to about 60 Gray. In a specific embodiment, the cells are treated with 1500 cGy ionizing radiation to produce feeder cells. Methods of irradiating feeder cells can be found in, e.g., U.S. Application Publication No. 2006/0223183. In a specific embodiment, the feeder cells are cryopreserved prior to irradiation. In another specific embodiment, a plurality of human fibroblasts are irradiated with from about 1 Gray to about 60 Gray, e.g., about 1.5 Gray, ionizing radiation to produce a plurality of feeder cells.

In one embodiment, the feeder cells and hematopoietic cells are cultured in a bioreactor, e.g., a bioreactor substantially as described elsewhere herein. In a specific embodiment of the bioreactor, the hematopoietic cells and feeder cells are co-cultured, that is, such that a plurality of the hematopoietic cells are in physical contact with a plurality of the feeder cells. In another embodiment, the hematopoietic cells are cultured separately, wherein culture medium from the feeder cells is circulated to the hematopoietic cells. In a specific embodiment, the hematopoietic cells expanded using a feeder layer are hematopoietic cells that were expanded with an immunomodulatory compound prior to culture with or contact with said feeder layer.

5.2.4. Adherent Placental Stem Cells as Feeder Cells

As noted above, adherent placental stem cells can be used as feeder cells in the methods provided herein. Adherent placental stem cells are described in, and are obtainable by the methods disclosed in, U.S. Pat. Nos. 7,045,148 and 7,255,879, and in United States Patent Application Publication Nos. 2007/0275362, and 2008/0032401, the disclosures of each of which are incorporated herein in their entireties.

Adherent placental stem cells useful in the methods provided herein express a plurality of markers that can be used to identify and/or isolate the stem cells, or populations of cells that comprise the stem cells. The adherent placental stem cells, and stem cell populations thereof (that is, two or more placental stem cells) include stem cells and stem cell-containing cell populations obtained directly from the placenta, or any part thereof (e.g., amnion, chorion, placental cotyledons, and the like). Adherent placental stem cell populations also includes populations of (that is, two or more) placental stem cells in culture, and a population in a container, e.g., a bag. The adherent placental stem cells described herein as usable as feeder layers for hematopoietic cell expansion are not trophoblasts, cytotrophoblasts, embryonic stem cells or embryonic germ cells.

The adherent placental stem cells described herein generally express the markers CD73, CD105, CD200, HLA-G, and/or OCT-4, and do not express CD34, CD38, or CD45. Placental stem cells can also express HLA-ABC (MHC-1) and HLA-DR. These markers can be used to identify adherent placental stem cells, and to distinguish adherent placental stem cells from other stem cell types. Because the placental stem cells can express CD73 and CD105, they can have mesenchymal stem cell-like characteristics. However, because the placental stem cells can express CD200 and HLA-G, a fetal-specific marker, they can be distinguished from mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stem cells, which express neither CD200 nor HLA-G. In the same manner, the lack of expression of CD34, CD38 and/or CD45 identifies the placental stem cells as non-hematopoietic stem cells.

Thus, in one embodiment, the feeder cells are isolated adherent placental stem cells that are CD200⁺ or HLA-G⁺. In another embodiment, the feeder cells are a population of cells comprising, e.g., that is enriched for, adherent CD200⁺, HLA-G⁺ placental stem cells. In another embodiment, the feeder cells are isolated adherent placental stem cells that are CD73⁺, CD105⁺, and CD200⁺. In another embodiment, the feeder cells are an isolated population of cells comprising, e.g., that is enriched for, adherent CD73⁺, CD105⁺, CD200⁺ placental stem cells. In another embodiment, the feeder cells are isolated adherent placental stem cells that are CD200⁺ and OCT-4⁺. In another embodiment, the feeder cells are an isolated population of cells comprising, e.g., that is enriched for, adherent CD200⁺, OCT-4⁺ placental stem cells. In another embodiment, the feeder cells are isolated adherent placental stem cells that are CD73⁺, CD105⁺ and HLA-G⁺. In another embodiment, the feeder cells are an isolated population of cells comprising, e.g., that is enriched for, adherent CD73⁺, CD105⁺ and HLA-G⁺ placental stem cells. In another embodiment, the feeder cells are isolated adherent placental stem cells that are CD73⁺ and CD105⁺ and which facilitate the formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said stem cells when said population is cultured under conditions that allow formation of embryoid-like bodies. In another embodiment, the feeder cells are a population of isolated cells comprising, e.g., that is enriched for, adherent CD73⁺, CD105⁺ placental stem cells, wherein said population forms one or more embryoid-like bodies under conditions that allow formation of embryoid-like bodies. In another embodiment, the feeder cells are isolated adherent placental stem cells that are OCT-4⁺ and which facilitate formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said stem cell when cultured under conditions that allow formation of embryoid-like bodies. In another embodiment, the feeder cells are a population of isolated cells comprising, e.g., that is enriched for, adherent OCT-4⁺ placental stem cells, wherein said population forms one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.

In another embodiment, the feeder cells comprise isolated adherent placental stem cells that are CD10⁺, CD34⁻, CD105⁺, and CD200⁺. In a specific embodiment of the above embodiments, said stem cells are additionally CD90⁺ and CD45⁻. In another embodiment, the feeder cells are isolated adherent placental stem cells that are HLA-A,B,C⁻, CD45⁻, CD133⁻ and CD34⁻. In another embodiment, the feeder cells are isolated adherent placental stem cell that is CD10⁺, CD13⁺, CD33⁺, CD45⁻, CD117⁻ and CD133⁻. In another embodiment, the feeder cells are isolated adherent placental stem cells that are CD10⁻, CD33⁻, CD44⁺, CD45⁻, and CD117⁻. In another embodiment, the feeder cells are isolated adherent placental stem cells that are HLA A,B,C⁻, CD45⁻, CD34⁻, CD133⁻, positive for CD10, CD13, CD38, CD44, CD90, CD105, CD200 and/or HLA-G, and/or negative for CD117. In another embodiment, feeder cells are isolated adherent placental stem cells that are CD200⁺ and CD10⁺, as determined by antibody binding, and CD117⁻, as determined by both antibody binding and RT-PCR. In another embodiment, the feeder cells are isolated adherent placental stem cells that are CD10⁺, CD29⁻, CD54⁺, CD200⁺, HLA-G⁺, HLA class I⁺ and β-2-microglobulin⁺.

Adherent placental stem cells used as feeder layers can be freshly isolated from the placenta, or can have been, for example, passaged at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.

Adherent placental stem cells can be used as feeder cells, for example, by contacting adherent placental stem cells with mitomycin C, or irradiating the cells, for a time sufficient to detectably suppress proliferation of the cells, and plating the cells, e.g., on a tissue culture surface, at about 5,000 cells per cm². The adherent placental stem cells are allowed to attach to the surface, e.g., for 10 minutes to 60 minutes. The plated, attached adherent placental stem cells are then ready to be inoculated with hematopoietic cells for hematopoietic cell culture and expansion.

5.3. Production of Erythrocytes from Hematopoietic Cells

Production of erythrocytes from hematopoietic cells, e.g., the expanded hematopoietic cells described above, is preferably performed in a bioreactor, e.g., the bioreactor exemplified elsewhere herein.

Differentiation of hematopoietic cells into erythrocytes and/or polychromatophilic erythrocytes can be accomplished by contacting the hematopoietic cells with one or more erythrogenic cytokines and/or growth factors. In one embodiment, hematopoietic cells are contacted with one or more of stem cell factor (SCF), erythropoietin (Epo), insulin-like growth factor (IGF-1), FMS-like tyrosine kinase 3 ligand (Flt3-L), and thrombopoietin (Tpo) in an amount and for a time sufficient to cause the differentiation of a plurality of the hematopoietic cells to erythrocytes and/or polychromatophilic erythrocytes. In various specific embodiments, at least 50%, 55%, 60%, 65%, 70%. 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the hematopoietic cells are differentiated to erythrocytes and/or polychromatophilic erythrocytes.

In a specific embodiment, the hematopoietic cells are contacted with cytokines, e.g., SCF, IGF-1 and erythropoietin in an amount and for a time sufficient to cause differentiation of a plurality of the hematopoietic cells into erythrocytes and/or polychromatophilic erythrocytes. In certain embodiments, the hematopoietic cells are contacted with one or more cytokines or growth factors in an amount and for a time sufficient to cause differentiation of a plurality of the hematopoietic cells into erythrocytes and polychromatophilic erythrocytes (also known as reticulocytes, blood reticulocytes, diffusely basophilic erythrocytes, or marrow reticulocytes). In certain other embodiments, the hematopoietic cells are contacted with one or more cytokines or growth factors in an amount and for a time sufficient to cause differentiation of a plurality of the hematopoietic cells into a precursor of an erythrocyte, e.g., into colony-forming units-granulocyte, erythrocyte, monocyte; blast-forming units-erythrocyte; colony-forming units-erythrocyte, pronormoblasts, basophilic normoblasts, polychromatic normoblasts, orthochromic normoblasts, or polychromatic erythrocytes (reticulocytes).

The above cytokines can be used in any amount that causes differentiation a plurality of the hematopoietic cells into erythrocytes. Typical amounts of cytokines used, for hematopoietic cells expanded for about 7 days from a starting population of, e.g., about 1×10⁴ to 2×10⁴ hematopoietic cells per milliliter, are about 50 ng/mL SCF; about 3 units/mL Epo; 50 ng/mL IGF-1; 50 ng/mL Flt3-L; and/or 100 ng/mL Tpo in, e.g., RPMI medium. Differentiation of the hematopoietic cells in, e.g. SCF, IGF-1 and Epo can proceed for, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, typically about 14 days. Optionally, the differentiated cells can be collected, resuspended to about 5×10⁵ per milliliter in medium, e.g., RPMI medium comprising Epo (e.g., at about 3 U/mL), IGF-1 (e.g., at about 20 ng/ml), IL-11 (e.g., at about 20 ng/ml) and IL-3 (e.g., at about 50 ng/ml), and cultured for an additional 1, 2, 3, 4, 5, 6, 7, 8 9 or 10 days, typically about 6 days.

In one embodiment, differentiation of hematopoietic cells, e.g., the expanded hematopoietic cells described above, can be accomplished by culturing said cells in contact with an immunomodulatory compound, e.g., a TNF-α inhibitory compound as described above, for a time and in an amount sufficient to cause a detectable increase in the proliferation of the hematopoietic cells over a given time, compared to an equivalent number of hematopoietic cells not contacted with the immunomodulatory compound. See, e.g., U.S. Patent Application Publication No. 2003/0235909, the disclosure of which is incorporated herein by reference in its entirety.

Differentiation of the hematopoietic cells into erythrocytes can be assessed by detecting erythrocyte-specific markers, e.g., by flow cytometry. Erythrocyte-specific markers include, but are not limited to, CD36 and glycophorin A. Differentiation can also be assessed by visual inspection of the cells under a microscope. The presence of typical biconcave cells confirms the presence of erythrocytes. The presence of erythrocytes (including reticulocytes) can be confirmed using a stain for deoxyribonucleic acid (DNA), such as Hoechst 33342, TO-PRO®-3, or the like. Nucleated precursors to erythrocytes typically stain positive with a DNA-detecting stain, while erythrocytes and reticulocytes are typically negative. Differentiation of hematopoietic cells to erythrocytes can also be assessed by progressive loss of transferring receptor (CD71) expression and/or laser dye styryl staining during differentiation. Erythrocytes can also be tested for deformability using, e.g., an ektacytometer or diffractometer. See, e.g., Bessis M and Mohandas N, “A Diffractometric Method for the Measurement of Cellular Deformability,” Blood Cells 1:307 (1975); Mohandas N. et al., “Analysis of Factors Regulating Erythrocyte Deformability,” J. Clin. Invest. 66:563 (1980); Groner W et al., “New Optical Technique for Measuring Erythrocyte Deformability with the Ektacytometer,” Clin. Chem. 26:1435 (1980). Fully-differentiated erythrocytes have a mean corpuscular volume (MCV) of about 80 to about 108 fL (femtoliters); mean corpuscular hemoglobin (MCH) of about 17 to about 31 pg, and a mean corpuscular hemoglobin concentration (MCHC) of about 23% to about 36%.

5.4. Separation of Erythrocytes from Precursors

Erythrocytes produced by the methods described above are preferably separated from hematopoietic cells, and, in certain embodiments, from precursors of erythrocytes. Such separation can be effected, e.g., using antibodies to CD36 and/or glycophorin A. Separation can be achieved by known methods, e.g., antibody-mediated magnetic bead separation, fluorescence-activated cell sorting, passage of the cells across a surface or column comprising antibodies to CD36 and/or glycophorin A, or the like. In another embodiment, erythrocyte separation is achieved by deoxygenating the culture medium comprising the erythrocytes, followed by magnetic separation of deoxygenated erythrocytes from other cells.

Erythrocytes can be continuously separated from a population of cells, e.g., from a second expanded hematopoietic cell population as described above, or can be separated at intervals. In certain embodiments, for example, isolation of erythrocytes is performed, e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or more, or when one or more culture condition criteria are met, e.g., achievement in the culture of a particular cell density; achievement in the culture of a particular number of cells per milliliter expressing certain erythrocyte markers, e.g., CD36 or glycophorin A; or the like. Separation of erythrocytes from a cell population is preferably performed using a bioreactor, as described below.

5.5. Bioreactor Production of Erythrocytes

In another aspect of the method of producing erythrocytes, hematopoietic cells are expanded using a bioreactor. In one embodiment, hematopoietic cells are expanded in a bioreactor in the presence of feeder cells. In another embodiment, hematopoietic cells are differentiated in a bioreactor. In a more preferred embodiment, the hematopoietic cells are expanded in a bioreactor, e.g., in the presence of feeder cells, then differentiated in the bioreactor. The bioreactor in which the hematopoietic cells are differentiated can be the same bioreactor in which the hematopoietic cells are expanded, or can be a separate bioreactor. In another embodiment, the bioreactor is constructed to facilitate expansion of the hematopoietic cells entirely in the bioreactor. In another embodiment, the bioreactor is constructed to allow expansion of hematopoietic cells in conjunction with feeder cells. In another embodiment, the bioreactor is constructed so as to physically separate the hematopoietic cells from the feeder cells. In another embodiment, the bioreactor is constructed to allow the hematopoietic cells and feeder cells to contact one another.

In another embodiment, the bioreactor is constructed to allow continuous flow of cells in media, enabling the continuous separation of differentiated erythrocytes from remaining cells in the bioreactor. The continuous flow and cell separation allows for the bioreactor to be constructed in a substantially smaller volume than would bioreactors using batch methods of producing erythrocytes. In another embodiment, the bioreactor is constructed to allow periodic, e.g., non-continuous flow of cells in media, enabling the periodic separation of differentiated erythrocytes from remaining cells in the bioreactor. The periodic flow and cell separation preferably allows for the bioreactor to be constructed in a substantially smaller volume than would bioreactors using batch methods of producing erythrocytes. In specific embodiments, isolation of erythrocytes is performed, e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or more. In another specific embodiment, isolation of erythrocytes is performed periodically when one or more culture condition criteria are met, e.g., achievement in the culture of a particular cell density; achievement in the culture of a particular number of cells per milliliter expressing certain erythrocyte markers, e.g., CD36 or glycophorin A; or the like.

In certain embodiments, the bioreactor is disposable.

In one embodiment, the bioreactor comprises a culturing element and a cell separation element. In another embodiment, the bioreactor comprises a medium gas provision element. In another embodiment, the bioreactor comprises a cell factor element comprising one or more bioactive compounds. In another embodiment, the elements of the bioreactor are modular; e.g., separable from each other and/or independently usable. In one embodiment, the capacity of the bioreactor is about 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, or about 900 mL, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45 or 50 liters. In another embodiment, the bioreactor, including all components, occupies about 47 cubic feet or less. In another embodiment, the bioreactor is capable of culturing up to about 10¹⁰, 10¹¹, or about 10¹² cells, e.g., hematopoietic cells.

In one embodiment, the culturing element comprises a compartment able to receive culture medium, e.g., culture medium comprising hematopoietic cells or culture medium comprising feeder cells. The culturing element comprises a port that allows for the introduction of media and/or hematopoietic cells for culture. Such a port can be any art-acceptable port for such devices, e.g., a Luer-lock seal port. The culturing element also comprises one or more ports for the passage of media to the cell separation element. The culturing element optionally further comprises a port for the introduction of bioactive compounds into the interior of the culturing element, e.g., a port that facilitates connection of the cell factor element to the culturing element. In a specific embodiment, hematopoietic cells, including differentiating hematopoietic cells, in the culturing element are continuously circulated in medium to a cell separation element (see below) to isolate erythrocytes and/or polychromatophilic erythrocytes and/or other erythrocyte precursors.

The culturing element, in a specific embodiment, comprises a plurality of interior surfaces or structures suitable for the culture of feeder cells, e.g., stromal cells or adherent placental stem cells. Such surfaces can be, e.g., tubes, cylinders, hollow fibers, a porous substrate, or the like. The surfaces can be constructed of any material suitable for the culture of cells, e.g., tissue culture plastic, flexible pharmaceutical grade plastic, hydroxyapatite, polylactic acid (PLA), polyglycolic acid copolymer (PLGA), polyurethane, polyhydroxyethyl methacrylate, or the like. Hollow fibers typically range from about 100 μm to about 1000 μm in diameter, and typically comprise pores that allow passage of molecules no more than about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 125 kDa, 150 kDa, 175 kDa, 200 kDa, 150 kDa, 300 kDa, 350 kDa, 400 kDa, 450 or 500 kDa.

In one embodiment, the surfaces on which the feeder cells are cultured physically separate the feeder cells from the hematopoietic cells. For example, the surfaces can be hollow fibers in the lumen of which the feeder cells are grown, while hematopoietic cells are cultured in media surrounding the fibers. The surfaces can also comprise a set of stacked membranes that separate two medium compartments, one for the feeder layers and another for the hematopoietic cells. In another embodiment, the surface on which the feeder cells are grown allows for the direct contact between feeder cells and hematopoietic cells.

The cell separation element comprises at least one port for receiving medium, comprising cells, from the culturing element. The cell separation element comprises one or more components that facilitate or enable the separation of at least one type of cell, e.g., erythrocytes, from cells in medium from the culture element. Such separation can be effected, e.g., using antibodies to CD36 and/or glycophorin A. Separation can be achieved by known methods, e.g., antibody-mediated magnetic bead separation, fluorescence-activated cell sorting, passage of the cells across a surface or column comprising antibodies to CD36 and/or glycophorin A, or the like. In a specific embodiment, the cell separation element is connected to the cell culturing element, and medium comprising hematopoietic cells, differentiating hematopoietic cells and erythrocytes is continually passed through the cell separation element so as to continually remove cells, e.g., erythrocytes from the medium.

In another embodiment, erythrocyte separation is achieved by deoxygenating culture medium comprising the erythrocytes, followed by magnetic attraction of deoxygenated erythrocytes, e.g., to a surface or other point of collection.

In another embodiment, the bioreactor comprises a cell separation element. The cell separation element can comprise one or more components that enable the separation of one or more non-erythrocytic cells (e.g., undifferentiated or non-terminally differentiated hematopoietic cells) from erythrocytes in the medium. In certain embodiments, the cell separation element is able to calculate an approximate number of erythrocytes generated, or is able to alert a user that a sufficient number of erythrocytes has been generated to constitute a unit, according to preset user parameters.

The bioreactor, in another embodiment, further comprises a gas provision element that provides appropriate gases to the culture environment, e.g., contacts the culture medium with a mixture of 80% air, 15% O₂ and 5% CO₂, 5% CO₂ in air, or the like. In another embodiment, the bioreactor comprises a temperature element that maintains the medium, the bioreactor, or both at a substantially constant temperature, e.g., about 35° C. to about 39° C., or about 37° C. In another embodiment, the bioreactor comprises a pH monitoring element that maintains the medium at a constant pH, e.g., about pH 7.2 to about pH 7.6, or about pH 7.4. In specific embodiments, the temperature element and/or pH monitoring element comprises a warning that activates when temperature and/or pH exceed or fall below set parameters. In other specific embodiments, the temperature element and/or pH monitoring element are capable of correcting out-of-range temperature and/or pH.

In a specific embodiment, the bioreactor comprises a cell separation element and a gas provision element that provides gases to the culture environment, whereby the gas provision element enables the partial or complete deoxygenation of erythrocytes, enabling erythrocyte separation based on the magnetic properties of the hemoglobin contained therein. In a more specific aspect, the bioreactor comprises an element that allows for the regular, or iterative, deoxygenation of erythrocytes produced in the bioreactor, to facilitate magnetic collection of the erythrocytes.

In another embodiment, the function of the bioreactor is automated, e.g., controlled by a computer. The computer can be, for example, a desktop personal computer, a laptop computer, a Handspring, PALM® or similar handheld device; a minicomputer, mainframe computer, or the like.

5.6. Erythrocyte Units Produced From Hematopoietic Cells

Erythrocyte units produced according to the methods detailed above can comprise erythrocytes in any useful number or combination of genetic backgrounds.

In various embodiments, erythrocyte units produced by the methods provided herein comprise at least about, at most about, or about 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 1×10¹² erythrocytes. In various other embodiments, the erythrocyte units comprise at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% completely-differentiated erythrocytes. In various other embodiments, the erythrocyte units comprise less than 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% erythrocyte precursors of any kind. In certain embodiments, the erythrocyte units produced by the methods described herein comprise less than about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 14%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6% or about 5% reticulocytes, or other non-erythrocytic hematopoietic cells. In another embodiment, the unit comprises erythrocytes from hematopoietic cells from a single individual. In another embodiment, the unit comprises erythrocytes differentiated from hematopoietic cells from a plurality of individuals. In another embodiment, the unit comprises erythrocytes from hematopoietic cells from matched human placental perfusate and cord blood. In another embodiment, substantially all (e.g., greater than 99%) of the erythrocytes in a unit of erythrocytes are type O. In another embodiment, substantially all (e.g., greater than 99%) of the erythrocytes in a unit of erythrocytes are type A. In another embodiment, substantially all (e.g., greater than 99%) of the erythrocytes in a unit of erythrocytes are type B. In another embodiment, substantially all (e.g., greater than 99%) of the erythrocytes in a unit of erythrocytes are type AB. In another embodiment, substantially all (e.g., greater than 99%) of the erythrocytes in a unit of erythrocytes are Rh positive. In another embodiment, substantially all (e.g., greater than 99%) of the erythrocytes in a unit of erythrocytes are Rh negative.

Naturally-occurring erythrocytes possess certain characteristics that allow the flow of blood through capillaries. For example, erythrocytes in the aggregate produce non-Newtonian flow behavior, e.g., the viscosity of blood is highly dependent upon shear rates. Normal erythrocytes are deformable and able to build up aggregates/rouleaux. The deformability of erythrocytes appears to be related to their lifespan in the blood, about 100-120 days; removal of erythrocytes from the blood appears to be related to loss of deformability. Normal aggregability of erythrocytes facilitates the cells' flow through the capillaries, while abnormally increased or decreased aggregability decreases flow. Thus, in preferred embodiments, units of erythrocytes produced by the methods disclosed herein are assayed as a part of quality control, e.g., for one or more characteristics of naturally-occurring erythrocytes. In certain embodiments, samples of erythrocytes produced by the methods disclosed herein are suspended in natural or artificial plasma and tested for one or more of viscosity, viscoelasticity, relaxation time, deformability, aggregability, blood/erythrocyte suspension yield stress, and mechanical fragility, using normal blood or normal erythrocytes as a control or comparator. In certain other embodiments, samples of erythrocytes produced as described herein are assayed for oxygen carrying capacity and oxygen release capacity, using normal blood or an equivalent number of naturally-occurring erythrocytes as a control.

6. EXAMPLES 6.1. Example 1: Phenotypic Characterization of CD34⁺ Cells from Human Placental Perfusate (HPP) and Umbilical Cord Blood (UCB)

Umbilical cord blood (UCB) was removed from postpartum placentas under informed consent. The exsanguinated placentas were then perfused to generate HPP, as described in U.S. Pat. No. 7,045,148, the disclosure of which is incorporated herein by reference in its entirety. After removal of red blood cells (RBCs) the total nucleated cells (TNCs) were collected and frozen. This method typically resulted in the collection of about 1-2.5×10⁹ TNCs, compared to around 500 million TNC isolated from UCB.

Flow cytometric analysis of the TNC isolated from exsanguinated placentas indicates a high percentage of CD34⁺ cell population as compared to conventional umbilical cord blood (UCB) generated cellular product. TNC from HPP, collected as above, contains about 2-6% CD34⁺ cells, compared to about 0.3-1% of the TNC in UCB.

The flow cytometric analysis of the TNC isolated from HPP indicates that a high percentage of the CD34⁺ cell population is CD45⁻ (FIG. 1).

CD34⁺ cells from HPP were plated in a colony-forming unit assay, and the ratio (%) of the burst forming unit-erythroid (BFU-E) to the colony forming unit-erythroid (CFU-E) was determined, as well as the number of colony-forming unit-granulocyte, macrophage (CFU-GM) and the number of colony-forming unit-granulocyte, erythrocyte, monocyte (CFU-GEMM) (Table 1). The clonogenicity was also assessed (Table 1).

TABLE 1 Cell CFU- Sample Purity BFU-E/CFU-E CFU-GM GEMM Clonogenicity Donor 1 88% 50.1% 49.5% 0.4% 23.1% Donor 2 92% 54.1% 44.1% 1.7% 26.1% Donor 3 94% 32.7% 60.6% 6.7% 19.7%

The colony-forming unit assay was performed according to the manufacturer's protocol (StemCell Technologies, Inc.). In brief, CD34⁺ cell suspensions were placed into a methylcellulose medium supplemented with stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), interleukin 6 (IL-6) and erythropoietin (Epo) at 100 cells/plate, 300 cells/plate and 1000 cells/plate. For each cell density, a triplicate assay was performed followed by incubation for 2 to 3 weeks. Colony evaluation and enumeration were performed using light microscopy.

In a separate experiment, the ratio (BFU-E)/(CFU-E) for CD34⁺ cells from HPP and UCB was 46% and 30%, respectively (based on the average value for three donors).

These results suggest that HPP-derived cells contain a higher number of CD34⁺ cells with increased erythrogenic activity relative to UCB-derived stem cells.

6.2. Example 2: Expansion of CD34⁺ Hematopoietic Cell Populations

The CD34⁺ cell content of human umbilical cord blood (UCB) units is often not sufficient to provide for hematopoietic cell transplants in adult patients. Ex-vivo expansion of CD34⁺ cells from UCB is one approach to overcome this CD34⁺ cell dose limitation. This Example demonstrates expansion of CD34⁺ cells using a specific immunomodulatory drug, 4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (referred to in this Example as pomalidomide).

The ability of pomalidomide to enhance the expansion of human UCB derived CD34⁺ cells in a short-term serum-free, cytokine supplemented culture system was evaluated. CD34⁺ progenitor cells were enriched from cryopreserved UCB units to >90% purity and seeded (10⁴ CD34⁺ cells) in 1 mL of growth medium, which consists of IMDM plus serum substitute BIT (BSA, recombinant human insulin and transferrin, 20%), in the presence of SCF (50 ng/mL), Flt-3 ligand (50 ng/mL), and IL-3 (10 ng/mL). Pomalidomide, dissolved in DMSO, was supplemented at 2.7 μg/mL. The culture was incubated at 37° C., 5% CO₂ for 12 days, with fresh medium added at day 7. Pomalidomide-free cultures with or without DMSO (0.05% v/v) were used as controls.

In one experiment, pomalidomide supplementation resulted in significantly higher CD34⁺ expression in the expanded population without impacting total nucleated cell expansion (200-350 fold). CD34⁺ phenotype in the pomalidomide-expanded population was 40-60%, compared with 10-30% in the control. Additionally, pomalidomide appeared to down-regulate CD38 expression on cultured cells. Pomalidomide-expanded CD34⁺ cells were primarily CD38 negative (95%) and expressed lower levels of CD133 (15% vs. 40% in the control). Pomalidomide-expanded CD34⁺ cells demonstrated substantial improvement in cumulative colony forming units relative to expanded controls. In another, similar, experiment, pomalidomide supplementation was confirmed to result in significantly higher CD34⁺ expression in the expanded population without impacting total nucleated cell expansion (200-350 fold; FIG. 2A). CD34⁺ phenotype in the pomalidomide-expanded population was 40-60%, compared with 10-30% in the control (FIG. 2B). Additionally, pomalidomide appeared to down-regulate CD38 expression on cultured cells.

Pomalidomide-expanded CD34⁺ cells were primarily CD38 negative (97%) and expressed lower levels of CD133 (11.5% vs. 32.3% in the control). Pomalidomide-expanded CD34⁺ cells demonstrated substantial improvement in cumulative colony forming units relative to expanded controls.

The pomalidomide-based CD34⁺ expansion process was scaled up to demonstrate the production of a larger number of CD34⁺ cells. CD34⁺ cells were seeded in 10⁴/mL pomalidomide-supplemented medium in a flexible, gas-permeable fluorocarbon culture bag (American Fluoroseal). After 7 days of incubation, the culture was centrifuged and exchanged with fresh pomalidomide-supplemented medium at three times the initial volume. By day 12, TNC and CD34⁺ expansion were 350 (range: 250-700) and 200 (range: 100-450) fold, respectively (FIG. 3A and FIG. 3B, respectively). Viability was 86% (range: 80-90%) by trypan blue. A total of 20 million CD34⁺ cells were harvested. These results demonstrate that pomalidomide significantly enhanced the ex-vivo expansion of placental derived CD34⁺ progenitors and that the process can produce a sufficient amount of CD34⁺ cells for erythrocytic differentiation (FIG. 3C).

6.3. Example 3: Feeder Cell-Free Expansion of CD34⁺ Precursors and Differentiation into Erythrocytes

HPP and UCB cells were generally purified using Ficoll to obtain total nucleated cells (TNCs). TNCs were then used to isolate CD34⁺ cells using anti-CD34 beads and RoboSep following the protocol provided by the manufacturer (StemCell Technologies, Inc.). If greater than 80% cell purity was not achieved, further cell sorting was performed using FACSAria to achieve a purity greater than 90%. In this experiment, CD34⁺ cells were isolated with about 92% purity.

In vitro expansion of CD34⁺ cells (about 1×10⁴ CD34⁺ cells/mL) was performed in either Iscove's Modified Dulbecco's Medium (IMDM; GIBCO, Grand Island, N.Y.) or RPMI Medium (Sigma-Aldrich) containing Flt-3L (50 ng/mL), SCF (100 ng/mL) and Tpo (100 ng/mL) from day 0 to day 7, and continued thereafter in IMDM or RPMI medium containing SCF (50 ng/mL), Epo (3 units/mL) and IGF-1 (50 ng/mL) from day 8 to day 14. Total cell numbers were counted at 8, 9, 12, 15 and 18 days. At day 18 TNC expansion was 300 fold and cell viability was 90%. At day 14, the cells were suspended at 2×10⁴ to 5×10⁵ cells/mL in IMDM or RPMI medium containing 3 units/mL Epo, 50 ng/mL IGF-1, 20 ng/mL IL-11 (interleukin 11), and 50 ng/mL IL-3 (interleukin 3) (R&D Systems) and cultured to day 21. FIGS. 4A and 4B show the ex vivo expansion of CD34⁺ cells from HPP (FIG. 4A) and UCB (FIG. 4B) in cytokine supplemented RPMI Medium.

6.4. Example 4: Expansion of Hematopoietic Cell Populations Using Feeder Layers

This Example explains the use of adherent placental stem cells in co-culturing system using adherent placental stem cells as a feeder layer.

To induce erythrocytic differentiation of the expanded CD34⁺ cells, 5×10⁷ cells were resuspended at 1 to 2×10⁴/ml and cultured in RPMI Medium containing 50 ng/mL Flt3-L, 100 ng/mL Tpo, and 100 ng/mL SCF (R&D Systems) for 7 days. The cells collected on day 7 were resuspended at 5×10⁴ to 1×10⁵/mL in RPMI medium containing 50 ng/mL SCF, 3 units/mL Epo, and 50 ng/mL IGF-1 (R&D Systems) and cultured up to 14 days. To further differentiate expanded CD34⁺ cells, the expanded cells collected on day 14 were resuspended at 2×10⁴ to 5×10⁵/mL in RPMI medium containing 3 units/mL Epo, 50 ng/mL IGF-1 with the presence of adherent placental stem cells treated with mitomycin C. The adherent placental stem cells were plated at about 5,000 cells per cm² and allowed to attach to the tissue culture surface. Aliquots of cultures of CD34⁺ cells were taken at different time points for morphological analysis by Giemsa staining (FIGS. 5A-5D). Morphological analysis by Giemsa staining indicated that a percentage of CD34⁺ expanded cells underwent full maturation into polychromatophilic erythrocyte/erythrocytes (FIG. 5D). In a subsequent experiment, 11.8% of HPP CD34⁺ cells fully differentiated, and 2.37% of UCB CD34⁺ cells fully differentiated, into polychromatophilic erythrocyte/erythrocytes as determined by staining with TO-PRO®-3, a nuclear stain that detects DNA.

Adherent placental stem cells usable as feeder cells can be obtained by mechanical disruption and enzymatic digestion of placenta as follows. Approximately 1 g tissue samples are incubated in a shaker in 1 mg/mL collagenase 1A for 1 hour at 37° C. at 100 RPM followed by incubation with 0.25% trypsin for 30 minutes at 37° C. at 100 RPM. The digested tissue is washed three times with culture medium prior to transferring washed cells into 75 cm² tissue culture flasks. The stem cells are maintained in media consisting of 60% v/v Dulbecco's Modified Eagle's Medium-Low Glucose (DMEM-LG), 40% v/v MCDB-201, supplemented with 2% v/v fetal calf serum, 1× insulin-transferrin-selenium, lx lenolenic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹ M dexamethasone, 10⁻⁴M ascorbic acid 2-phosphate, 10 ng/ml epidermal growth factor, platelet derived-growth factor—BB (10 ng/ml), and 100 U penicillin/1000 U streptomycin, in a 5% CO₂ and humidified incubator at 37° C. Non-adherent cells are removed 12 to 24 hours post plating and half of the medium is exchanged every 3-4 days. Cells are subcultured using 0.25% trypsin-EDTA and replated at about 4×10³ cells/cm² for continued expansion.

6.5. Example 5: Method and Bioreactor for Generating Units of Erythrocytes

This Example provides a method of producing erythrocytes, and a bioreactor that enables the production of units of mature erythrocytes. In this particular example, the bioreactor enables the production of administrable units of erythrocytes using a five-step process. In the first step, hematopoietic cells, e.g., CD34⁺ cells, are isolated. In the second step, the CD34⁺ cells are expanded using an immunomodulatory compound, e.g., pomalidomide. In the third step, the CD34⁺ cells are expanded in the bioreactor exemplified herein, in a co-culture with adherent placental stem cells, in conjunction with removal of lineage-committed cells. Fourth, remaining uncommitted hematopoietic cells are differentiated to erythrocytes. Finally, in the fifth step, erythrocytes are isolated and collected into administrable units.

Steps 1 and 2, the isolation and initial expansion of hematopoietic cells, are accomplished as described in Examples 3 and 4, above.

Steps 3 and 4 are accomplished using a bioreactor. The bioreactor comprises a hollow fiber chamber (1) seeded with placental stem cells (2) and an element for gas provision to the medium (3). The bioreactor further comprises a coupled cell sorter/separator element (4) that allows for the continuous separation of committed hematopoietic cells, fully-differentiated erythrocytes, or both. The cell separation element can separate the cells from the hematopoietic cells using, e.g., magnetic cell separation or fluorescence-activated cell separation techniques.

To initiate cell culture, approximately 5×10⁷ hematopoietic cells, e.g., CD34⁺ hematopoietic cells, are inoculated into the bioreactor.

6.6. Example 6: Collection of Erythrocytes

This Example exemplifies several methods of the separation of erythrocytes from other lineage committed cells.

Method 1: Erythrocytes, e.g., erythrocytes collected from the cell separation element of the bioreactor exemplified herein, and hetastarch solution are mixed 3:1 (v:v) in a Baxter collection bag and placed in an upright position on a plasma extractor. Erythrocytes sediment after 50 to 70 minutes. Non-sedimented cells are forced out by the plasma extractor. Sedimented erythrocytes left in the bag can be further collected by centrifugation at 400 g for 10 minutes. After removing the supernatant, erythrocytes are resuspended in an appropriate amount of desired medium.

Method 2—Immunomagnetic separation: Glycophorin A⁺ cells, e.g., erythrocytes collected from the cell separation element of the bioreactor exemplified herein, are magnetically labeled with Glycophorin A (CD235a) MicroBeads (Miltenyi Biotech). The cell suspension is then loaded into a tube which is placed in the magnetic field of an EASYSEP® magnet. The magnetically labeled Glycophorin A⁺ cells are retained inside the tube, while the unlabeled cells are poured off the tube. After removal of the tube from the magnetic field, the magnetically retained Glycophorin A⁺ cells can be separated from the magnetic beads and resuspended in an appropriate amount of desired medium.

Method 3—Flow cytometry cell separation: Erythrocytes, e.g., erythrocytes collected from the cell separation element of the bioreactor exemplified herein, in 500 μL PBS/FBS with 1 μL Fc Block (1/500). 150 μL of the cell suspension is added to each well of a 96 well V-bottom dish. 50 μL 1° Ab Master Mix (the mix is a 1/25 dilution of each primary Ab in PBS/FBS) is added to the cells. One well is included with a combination of isotype controls for setting voltage, as well as one well for each of the primary Ab as single positive controls for setting compensation. The cells are incubated 60 min at 4° C., then centrifuged at 1500 RPM for two minutes. The supernatant is discarded. The wells are washed with 200 μL PBS/FBS to each well, and mixed by pipetting up and down. The cells are then immediately spun at 1500 RPM ×2 min; the supernatant is discarded. 150 μL of secondary Ab (i.e. Streptavidin-TC) Master Mix is added, and incubated 30 min at 4° C., followed by centrifugation at 1500 RPM for 5 minutes. The pellet is resuspended in 200-500 μL of PBS/FBS and transferred to 5 mL flow tubes. Cells are then separated using a flow cytometer.

Method 4: Medium comprising erythrocytes, in continuous flow between the cell culture element and cell separation element, is deoxygenated by reducing or turning off the supply of oxygen from the gas provision element, and turning on a magnet in the cell separation element. Medium is passed through the cell separation element for a sufficient time for the magnetic field of the magnet to collect erythrocytes to a surface in the cell separation element. Once a predetermined number of erythrocytes are collected, or collection has proceeded for a predetermined amount of time, the medium is reoxygenated, releasing the erythrocytes from the surface.

6.7. Example 7: Bioreactor for Producing Erythrocytes

This Example describes a bioreactor design that allows for improved production of erythrocytes from hematopoietic cells. The bioreactor comprises a culturing element that comprises hollow fibers in which hematopoietic cells and feeder cells are cultured. Hematopoietic cells, e.g., hematopoietic progenitor cells, are supplied in a bag at 5×10⁵ cells/dose, where one dose yields one unit of blood. The cells are expanded in the presence of IMDM medium containing 50 ng/mL SCF, 3 units/mL Epo, and 50 ng/mL IGF-1 added through a first port. Gas provision (5% CO₂ in air) occurs through a second port. The medium in which the hematopoietic cells are cultured is supplemented with pomalidomide at 2.7 μg/mL. The cells are cultured in contact with feeder cells (adherent placental stem cells) that have been seeded in the hollow fiber element of the bioreactor. During culturing, gas, medium metabolites and medium pH in the culturing element is monitored continuously, and are replenished or exchanged using a programmable control device as necessary. pH of the medium in the culturing element is maintained at approximately 7 and the culture temperature is maintained at about 37° C. Lineage-committed cells (i.e., differentiated cells) are continuously separated and recovered from the culture medium using a cell separation element. The bioreactor is equipped with an independent power supply to enable operation at a remote site, e.g., a site separate from a site at which hematopoietic cells are initially obtained.

The present disclosure, including devices, compositions and methods, is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention. 

What is claimed is:
 1. A method of producing erythrocytes, comprising (a) expanding a plurality of hematopoietic cells in the absence of feeder cells, optionally in contact with an immunomodulatory compound, wherein the immunomodulatory compound increases the number of hematopoietic cells compared to a plurality of hematopoietic cells not in contact with the immunomodulatory compound, to produce a first expanded hematopoietic cell population; (b) expanding the first expanded hematopoietic cell population in the presence of a plurality of feeder cells to produce a second expanded hematopoietic cell population; (c) contacting said second expanded hematopoietic cell population with one or more factors that cause differentiation of hematopoietic cells in said second expanded hematopoietic cell population into erythrocytes; and (d) isolating said erythrocytes from said second expanded hematopoietic cell population.
 2. The method of claim 1, wherein said hematopoietic cells are CD34⁺.
 3. The method of claim 1, wherein said hematopoietic cells are Thy-1⁺, CXCR4⁺, CD133⁺ or KDR⁺.
 4. The method of claim 2, wherein said hematopoietic cells are CD45⁻.
 5. The method of claim 1, wherein said hematopoietic cells are HLA-DR⁻, CD71⁻, CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD16⁻, CD24⁻, CD56⁻, CD66b⁻ and/or glycophorin A⁻.
 6. The method of claim 1, wherein said hematopoietic cells are obtained from cord blood, placental blood, peripheral blood, or bone marrow.
 7. The method of claim 1, wherein said hematopoietic cells are obtained from placental perfusate.
 8. The method of claim 1, wherein said hematopoietic cells are obtained from umbilical cord blood and placental perfusate.
 9. The method of claim 6, wherein said placental perfusate is obtained by passage of perfusion solution through only the vasculature of a placenta.
 10. The method of claim 7, wherein said placental perfusate is obtained by passage of perfusion solution through only the vasculature of a placenta.
 11. The method of claim 1, wherein said hematopoietic cells are human hematopoietic cells.
 12. The method of claim 1, wherein said feeder cells are from the same individual as said hematopoietic cells.
 13. The method of claim 1, wherein said feeder cells are from a different individual than said hematopoietic cells.
 14. The method of claim 12, wherein said feeder cells are adherent placental stem cells, bone marrow-derived mesenchymal stem cells, mesenchymal stem cells from peripheral blood, mesenchymal stem cells from cord blood, or stromal stem cells, or a combination of any of the foregoing.
 15. The method of claim 12, wherein said feeder cells are adherent placental stem cells.
 16. The method of claim 15, wherein said adherent placental stem cells are: CD200⁺ or HLA-G⁺; CD73⁺, CD105⁺, and CD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁺; CD73⁺ and CD105⁺ and facilitate the formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said stem cells when said population is cultured under conditions that allow formation of embryoid-like bodies; OCT-4⁺ and which facilitate formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said stem cell when cultured under conditions that allow formation of embryoid-like bodies; CD10⁺, CD34⁻, CD105⁺, and CD200⁺; HLA-A,B,C⁺, CD45⁻, CD133⁻ and CD34⁻; CD10⁺, CD13⁺, CD33⁺, CD45⁻, CD117⁻ and CD133⁻; CD10⁻, CD33⁻, CD44⁺, CD45⁻, and CD117⁻; HLA A,B,C⁺, CD45⁻, CD34⁻, CD133⁻; positive for CD10, CD13, CD38, CD44, CD90, CD105, CD200 and/or HLA-G, and/or negative for CD117; CD200⁺ and CD10⁺, as determined by antibody binding, and CD117⁻, as determined by both antibody binding and RT-PCR; or CD10⁺, CD29⁻, CD54⁺, CD200⁺, HLA-G⁺, HLA class I⁺ and β-2-microglobulin⁺.
 17. The method of claim 1, wherein a plurality of said hematopoietic cells is blood type A, blood type O, blood type AB, blood type O; is Rh positive or Rh negative; blood type M, blood type N, blood type S, or blood type s; blood type P1; blood type Lua, blood type Lub, or blood type Lu(a); blood type K (Kell), k (cellano), Kpa, Kpb, K(a+), Kp(a−b−) or K-k-Kp(a−b−); blood type Le(a−b−), Le(a+b−) or Le(a−b+); blood type Fy a, Fy b or Fy(a−b−); or blood type Jk(a−b−), Jk(a+b−), Jk(a−b+) or Jk(a+b+).
 18. The method of claim 17, wherein the hematopoietic cells are type O, Rh positive; type O, Rh negative; type A, Rh positive; type A, Rh negative; type B, Rh positive; type B, Rh negative; type AB, Rh positive or type AB, Rh negative. 